The calyx of Held synapse is a giant axosomatic synapse that acts as a fast relay in the sound localization circuit of the brainstem. In rodents it forms within a relatively brief period starting around the second postnatal day (P2). The relative timing of the formation of its pre- and the postsynaptic compartment are not yet known. By means of fluorescent immunohistochemistry in neonatal rats we therefore compared the developmental expression patterns of the vesicular glutamate transporter (VGLUT)-1 and the postsynaptic density scaffolding proteins Homer-1 and PSD-95 in the medial nucleus of the trapezoid body (MNTB). Before its formation, colocalized punctate staining of VGLUT-1 and Homer-1 or PSD-95 was observed on principal neurons, in agreement with earlier work showing that they are already innervated by fibers from the cochlear nucleus before the calyx forms. The expression of VGLUT-1 clusters within the nascent calyx of Held synapse preceded the expression of Homer-1 and PSD-95 clusters, as indicated by the presence of principal neurons with only a large contiguous cluster (LCC) of VGLUT-1 at P2-3, whereas no neurons with only an LCC for Homer-1 or PSD-95 were seen. At P3 the first principal neurons with both a pre- and a postsynaptic LCC were observed, and at P12 all principal neurons had both a pre- and a postsynaptic LCC. The relatively late appearance of Homer-1 and PSD-95 within the developing calyx of Held synapse suggests that they play a role in its stabilization and the recruitment of additional receptors to its postsynaptic density. J. Comp. Neurol. 518:4581–4590, 2010. © 2010 Wiley-Liss, Inc.